α-Synuclein and γ-Tubulin Cooperatively Regulate Activity-Evoked Presynaptic Microtubule Nucleation to Gate Dopamine...

α-Synuclein has long been implicated in the regulation of synaptic activity, but the molecular basis that underlies this function has been elusive. Here, we identify a microtubule (MT)-dependent mechanism through which α-synuclein regulates synaptic dopamine release. Using live imaging of cultured dopaminergic neurons, we visualize dynamic MTs at individual presynaptic boutons and show that neuronal activity triggers local γ-tubulin-dependent MT nucleation. We find that this nucleation is essential for interbouton synaptic vesicle (SV) transport and for sustained dopamine release during high activity. We further discover that α-synuclein acts as a positive regulator of presynaptic MT nucleation by binding directly to γ-tubulin and the α/β-tubulin heterodimer. Activity-evoked phosphorylation of α-synuclein at serine 129, a modification that accumulates in synucleinopathies and a molecular switch for α-synuclein binding to synaptic proteins, occurs in the region of α/m tubulin binding and is both necessary and sufficient for MT initiation. Our findings reveal a previously unrecognized, activity-dependent role for α-synuclein in the nucleation of axonal MTs that enables on-demand SV interbouton redistribution and dopamine release. This mechanism provides a novel molecular link between α-synuclein phosphorylation and MT-dependent modulation of dopamine release, offering insight into how its dysregulation may contribute to dopaminergic synaptic dysfunction, a central feature of synucleinopathies.